Involvement of the mural thrombus as a site of protease release and activation in human aortic aneurysms

Abstract

Acquired abdominal aortic aneurysms are usually associated with a mural thrombus through which blood continues to flow. Some early data suggest that aneurysmal evolution correlates with the biological activity of the thrombus. Our hypothesis was therefore that the thrombus could adsorb blood components and store, release, and participate in the activation of proteases involved in aneurysmal evolution. For this purpose, we have explored both the metalloproteinase and fibrinolytic systems in the thrombus and the wall of human aneurysms. We have first investigated blood clot formation and lysis in vitro. Spontaneous clotting induces a release of promatrix metalloproteinase (pro-MMP)-9 into the serum that was fourfold higher than in paired control plasma (P < 0.001). Fibrinolysis progressively released more MMP-9 in a time-dependent manner (P < 0.01). After selective isolation, we demonstrated that polymorphonuclear leukocytes are the main source of MMP-9 release during clot formation. Protease content was then analyzed in 35 mural thrombi and walls of human abdominal aortic aneurysms sampled during surgical repair. In 15 aneurysms, the liquid phase at the interface between the thrombus and the wall was sampled separately. Both thrombus and wall contained MMP-2 and MMP-9 but the ratio MMP-9/MMP-2 was higher in the thrombus than in the wall. The liquid interface also contained active MMP-9. Immunohistochemistry of the thrombus confirmed these findings, showing the presence of polymorphonuclear leukocytes at the luminal pole of the thrombus, co-localizing with MMP-9 storage. In contrast, MMP-3 and MMP-7 were only present in the aneurysmal wall. Plasminogen was present in the mural thrombus but plasmin activity was present in both thrombus and wall. In the liquid interface, plasmin-alpha(2)-anti-plasmin complexes were detected demonstrating in vivo the activation of plasminogen. In contrast, u-PA and t-PA were detectable only in the wall, suggesting that plasminogen present in the thrombus could be activated by factors secreted by the arterial wall. This was demonstrated in vitro, in which co-incubation of thrombus and wall extracts generated plasmin in the presence of a fibrin matrix and activated MMPs. In conclusion, our study strongly suggests that the mural thrombus, by trapping polymorphonuclear leukocytes and adsorbing plasma components could act as a source of proteases in aneurysms that may play a critical role in enlargement and rupture.

Figure 1.

Gelatinase activities in plasma, serum, blood cell pellet, and clot. A: MMP-2 and MMP-9 activities, detected on zymograms (top), were measured in plasma (Pl), recalcified plasma (Pl + CaCl₂), and in serum as a function of time (MMP-9, middle; MMP-2, bottom). **, P < 0.01 as compared to zero point time; ^$$, P < 0.01; ^$$$, P < 0.001 as compared to plasma. B: Gelatinase activities were measured in extracts of blood cells, clot, and 7-day-lysed clot (top) and quantified by densitometry (MMP-9, middle; MMP-2, bottom). **, P < 0.01 as compared to cell pellet; ^$$, P < 0.01 as compared to nonlysed clot.

Figure 2.

Gelatinase activities in extracts of PMNs, monocytes/lymphocytes, and platelets. A: MMP-2 and MMP-9 activities, detected on zymograms. B: MMP-9 activity was quantified by densitometry. ***, P < 0.001 as compared to platelets;^$$, P < 0.01 as compared to monocytes/lymphocytes.

Figure 3.

Gelatinase activities in mural thrombus, liquid interface, and aneurysmal wall. MMP-2 and MMP-9 activities detected on zymograms (A) were further characterized by Western blot (B). MMP-9 (C) and MMP-2 (D) activities were quantified by densitometry and MMP-9/MMP-2 ratios (E) were calculated. **, P < 0.01 as compared to thrombus extracts.

Figure 4.

Immunochemistry of MMP-9 in mural thrombus. A and inset: Hematoxylin, phloxin, and safran staining showing the presence of PMNs in the luminal pole of the thrombus. B: MMP-9 staining in the luminal and abluminal pole of the thrombus and inset showing the co-localization of MMP-9 and PMNs in luminal pole. C: GPIIb/IIIa staining in the luminal and abluminal pole of the thrombus and inset showing a diffuse marking of GPIIb/IIIa corresponding to platelets in the luminal pole. PMN and platelet smears were used as positive controls for MMP-9 and GPIIb/IIIa immunostaining, respectively (data not shown). Original magnifications, ×100 (insets in A–C).

Figure 5.

Pg and Pn in the three compartments. Pg and Pn (A) and free α₂-anti-Pn (α₂-AP) and α₂-anti-Pn-Pn complexes (α₂-AP-Pn) (B) were detected on Western blots. Pg (C) and Pn quantities (D) were determined through their activity on a chromogenic substrate. **, P < 0.01 as compared to thrombus.

Figure 6.

Free Pg activators were localized in the wall. Pg activators were detected through the activation of Pg in fibrin-agar gels (A). t-PA (B) and u-PA (C) activities and uPA-PAI-1 complexes (D) were quantified by densitometry.

Figure 7.

In vitro interaction between thrombus and wall extracts. Thrombus/wall extract interactions were monitored in vitro in the absence (−) or presence (+) of a fibrin matrix. Pg from thrombus extracts was activated by the Pg activators contained in the wall extract on fibrin-coated wells (A, ♦, ▪, ▾: three representative experiments, initial rates: 364, 306, 226 fmol/min, respectively, and •: control experiments in BSA-coated wells). Co-incubated extracts were analyzed on gelatin zymography (B). Percentage of active MMP-9 (C) and MMP-2 (D) were calculated in the presence or absence of fibrin matrix. **, P < 0.01; ***, P < 0.001.